摘要
目的探讨应用RNA干扰技术同时沉默人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、Bcl-xl三个基因对喉鳞状细胞癌Hep-2细胞生长增殖的影响,并与单独沉默hTERT基因对细胞的生长增殖作用进行比较。方法构建表达hTERT mRNA特异的、含荧光素基因的shRNA(short hairpin RNA,短发夹RNA)真核表达质粒pshRNA1;根据hTERT、VEGF、Bcl-xl的cDNA序列构建串联表达3个shRNA序列的重组质粒pshRNA2。将上述质粒分别转染Hep-2细胞,MTT法观察Hep-2细胞增殖活性,流式细胞仪检测细胞凋亡,蛋白印迹法检验蛋白表达。结果pshRNA1、pshRNA2转染Hep-2细胞后,细胞增殖受到明显抑制,并诱导细胞凋亡。且pshRNA2对细胞的生长增殖抑制作用明显强于pshRNA1。pshRNA1转染细胞后hTERT蛋白表达下降,pshRNA2转染细胞后hTERT、VEGF、Bcl-xl蛋白表达均明显下降。结论与单独沉默hTERT相比,同时沉默hTERT、VEGF、Bcl-xl基因表现出更强大的抑制细胞增殖效应,多基因共沉默为喉癌的基因治疗提供了新思路。
OBJECTIVE To investigate the effect of RNA interference silencing the human telomerase reverse transcriptase （hTERT） , vascular endothelial growth factor （VEGF） and Bcl-xl mRNA simultaneously on the Hep-2 cells in larynx carcinoma cell line, and to compare the effect of silencing the hTERT gene alone. METHODS A recombinant plasmid containing 3 different short hairpin RNA （shTERT, shVEGF, shBcl-xl） segments termed pshRNA2 was constructed. Plasmid containing single shRNA （shTERT） termed pshRNA1 was also constructed. Cells were treated with these plasmids. Cell proliferation was examined using the MTT assay. The apoptosis of Hep-2 cells was detected by flow cytometer （FCM） . The protein expression was observed by western blotting. RESULTS After treatment with pshRNA1 and pshRNA2, the proliferation of Hep-2 cells was obviously inhibited . pshRNA2 exhibited a potent antitumor effect on Hep-2 cells, and could much more effectively induce cell apoptosis than pshRNAl. Our results demonstrated that a recombinant plasmid containing multiple shRNAs could effectively and simultaneously inhibit VEGF, hTERT, Bcl-xl mRNA and protein expression in the Hep-2 cells. CONCLUSION The plasmid containing the 3 different shRNAs is much more effective than a single shRNA, supplying a promising therapeutic modality in the gene therapy of human laryngeal carcinomas.
出处
《中国耳鼻咽喉头颈外科》
北大核心
2009年第3期123-127,共5页
Chinese Archives of Otolaryngology-Head and Neck Surgery
基金
国家自然科学基金面上项目(30471873
30672313
30740012)
湖北省科技攻关项目(2007AA302B08)
武汉市科技局国际合作项目(2006AA301C06)联合资助
关键词
RNA干扰
基因疗法
血管内皮生长因子
凋亡抑制蛋白质类
人端粒酶逆转录酶
RNA Interference
Gene Therapy
Vascular Endothelial Growth Factors
Inhibitor of Apoptosis Proteins
human telomerase reverse transcriptase
